Mutagenesis and Functional Characterization of theglnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

摘要

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation ofnif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization ofglnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P_II) were constructed. While P_II-Y51F showed a lower nitrogenase activity than that of wild type, a P_IIdeletion mutant showed very little nif expression. This effect of P_II on nif expression is apparently the result of a requirement of P_II for NifA activation, whose activity is regulated by NH₄ + in R. rubrum. The modification of glutamine synthetase (GS) in theseglnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P_II might exist inR. rubrum and regulate the modification of GS. P_II also appears to be involved in the regulation of DRAT activity, since an altered response to NH₄ + was found in a mutant expressing P_II-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH₄ + and darkness treatments.